Transgenic plant product development by conventional transformation and breeding efforts is a slow and unpredictable process. Gene targeting systems can overcome problems with expression variability, unpredictable impacts of random gene insertion on agronomic performance, and the large number of experiments that need to be conducted. Such systems can also provide approaches to manipulating endogenous genes. Of course, targeting system requires the ability to focus the recombination process to favor recovery of desired targeting events.
Recombination is the process by which DNA molecules are broken and rejoined, giving rise to new combinations. It is a key biological mechanism in mediating genetic diversity and DNA repair. Much research has focused on describing the process, since it is an integral biological phenomenon and as such, forms the basis of a number of practical applications ranging from molecular cloning to introduction of transgenes.
The natural cellular DNA repair and recombination machinery consists of a complex array of protein components interacting in a highly controlled manner to ensure that the fidelity of the genome is conserved throughout the many internal events or external stimuli experienced during each cell cycle. The ability to manipulate this machinery requires an understanding of how specific proteins are involved in the process, and how the genes that encode those proteins are regulated. Since the primary approaches to gene targeting involve recombinases, whether operating in their natural in vivo environment (as during normal recombination) or as part of schemes that involve pretreatment of substrates so as to associate DNA with a recombinase and increase the efficiency of targeting (e.g., double D-loop), there is a continuing need to isolate and characterize the genes for these molecules. Because many different protein components may be involved in gene targeting, the availability of host-specific genes and proteins could aid in avoiding possible problems of incompatibility associated with molecular interactions due to heterologous components.
A number of proteins involved in recombination have been isolated and the corresponding genes have been cloned, including RecA of E. coli, a key player in the recombination process. RecA catalyzes the pairing up of a DNA double helix and a homologous region of single-stranded DNA, and so initiates the exchange of strands between two recombining DNA molecules. It exhibits DNA-dependent ATPase activity, binding DNA more tightly when it has ATP bound than when it has ADP bound. RecA gene homologues in other organisms have been isolated, including RAD51 from human, mouse, corn, and yeast (Shinohara, A. et al., (1993) Nat. Genet. 4:239–243; PCT published Patent Application No. WO 99/41394-A1), and DMC1 from yeast, lily, and Arabidopsis (Klimyuk, V. I. and Jones, J. D., (1997) Plant J. 11:1–14). In fission yeast, a number of meiotic recombination genes have been identified by genetic complementation, including rec6 and rec12 (Lin, Y. and Smith, G. R. (1994) Genetics 136:769–779).
Sequences for the bacterial RecA recombinase and functional homologs from yeast and several animal species have been disclosed in various publicly accessible sequence databases. Numerous publications characterizing these recombinases exist (see, e.g., Kowalczykowski et al., Annu. Rev. Biochem., 63:991–1043 (1994)). Reports of the use of bacterial RecA in association with DNA sequences to manipulate homologous target DNA, including improvement of the efficiency of gene targeting in non-plant systems, have been published (see, e.g., PCT published Patent Application Nos. WO 87/01730 and WO 93/22443).
The catalysis of in vitro pairing and strand exchange between circular viral single strand DNA (“ss DNA”) and linear duplex DNA (“ds DNA”) by a RAD51 recombinase from S. cerevisiae has also been reported (see, e.g., Sung, Science, 265:1241–43 (1994); Kanaar, et al., Nature 391:335–338 (1998); Benson, et al., Nature 391:401–410 (1998)). To date, work with recombinase enzymes in plants, however, has been very limited. Accordingly, there is an ongoing need for the identification and characterization of the functional activities of recombinase enzymes which may offer improved and expanded methods for use in plant systems, particularly agriculturally important crop species.
Obtaining targeted knockouts of endogenous genes through introduction of homologous strands of DNA is a feat which has been achieved in mammalian cells several years ago. It is however an enormous challenge in plants, which is indicative of a lack of sufficient knowledge about homologous recombination in plant cells. Isolation and characterization of plant genes involved in recombination may help in overcoming the present obstacles.